Mechanism Of Baicalin Reverses The Resistance Of Multidrug-resistant Staphylococcus Saprophyticus From Francolins To Azithromycin

In recent years,antibiotic resistance severely threatened medicine and public health across the world,and the development of antibiotics is far less than the emergence of resistant bacteria.Staphylococcus saprophyticus is a major opportunistic and zoonotic pathogen.It also can restore many resistant genes and contribute to the spread of antibiotic resistance genes.It can cause many diseases such as human urinary system diseases,pneumonia,cow mastitis and avian ophthalmia.The threat of this bacteria is more serious in human than that in animals.In human medicine,azithromycin is often used to treat infections caused by S.saprophyticus,and the drug resistance is more serious.In veterinary medicine,fewer antibiotics are available for this infection.In order to prevent the spread and prevalence of resistant genes in bacteria derived from human and animals,resistance inhibitors are urgently required to develop.Many flavonoids have the potential to be resistance inhibitors.Baicalin is a flavonoid compound extracted from the roots of Scutellaria baicalensis Georgi and acts synergetic effects with many kinds of antibiotics.Azithromycin was also used to treat the infection of staphylococcus.And many bacteria were resistant to azithromycin.This study aimed to evaluate the effects and mechanisms of baicalin reversing the resistance of multidrug-resistant Staphylococcus saprophyticus to azithromycin.First,a multidrug-resistant Staphylococcus saprophyticus strain was isolated and identified from a francolin suffered from ophthalmia and named multidrug-resistant Staphylococcus saprophyticus-Liu-2016-Liyang,China-francolin（MDRSS）.Then the resistant profile of MDRSS was analyzed.The effect of baicalin reverses the resistance of MDRSS to antibiotics was investigated.The results indicated that baicalin reversed the resistance of MDRSS to macrolides antibiotics and sulfafurazole.Then the synergetic effects of baicalin and azithromycin against MDRSS in vivo and vitro were determined.The effects and mechanisms of baicalin reversing resistance were explored from two aspects of efflux pump and biofilm.The details are as follows:Test Ⅰ:Isolation,identification,and antimicrobial resistance of multidrug-resistant Staphylococcus saprophyticusA large number of francolins suffered from ophthalmia which induced the laying rate decline or even death on a francolins farm in Jiangsu province,China.Simultaneously,the symptom did not relieve when treated with a variety of antibiotics.The objective of the study was to isolate the pathogen from francolins suffering from ophthalmia and identify the antibiotic resistance profile.The secretions in the eyes were collected from francolins suffered from ophthalmia,and these samples were cultured with differentiation mediums.The suspected colony was confirmed with Gram stain and biochemical tests.The 16S rRNA and gap genes were amplified by PCR and PCR products were purified and sequenced.The animal regression test was conducted.Finally,the antibiotic resistance profile was detected by the Kirby-Bauer method.The results showed that the isolated bacteria grew classical morphology of staphylococcus in differentiation mediums.The Gram-positive cocci were seen under the microscope.It was positive for catalase and negative for coagulase.For the gap gene sequence,the best match was S.saprophyticus（>98%）.The case was replicated and the same pathogen was identified in the animal regression test.The pathogen was resistant to more than ten antibiotics and only sensitive to ampicillin,gentamicin,and clindamycin.These results indicated that multidrug-resistant S.saprophyticus was the pathogen that caused ophthalmia,it was named multidrug-resistant Staphylococcus saprophyticus-Liu-2016-Liyang,China-francolin（MDRSS）according to the nomenclature of microorganisms.Test Ⅱ:The mechanisms of MDRSS resistant to macrolides antibioticsThe aim of this study was to investigate the mechanisms of MDRSS to azithromycin.23S rRNA domain Ⅱ and Ⅴ,ermA,ermB,ermC,mphC,msrA and msrB genes were amplified by PCR.The products of 23S RNA were linked with T-vector and sequenced.The PCR products of other genes were sequenced and blasted.In order to confirm the existence of the efflux pump,the efflux inhibitor was added.The biofilm morphology was observed by SEM.The results showed that the 23S rRNA was not mutated.The mphC,msrA and msrB genes were amplified but the ermA,ermB and ermC genes were not amplified.When efflux inhibitor was added,the MIC of azithromycin against MDRSS declined 8-fold.MDRSS formed lots of biofilms.These results suggested that MDRSS was resistant to macrolide antibiotics because of mphC,msrA,msrB,and biofilm formation.Test Ⅲ:The effect of baicalin reverses the resistance of MDRSS to azithromycinThe aim of this study was to explore the roles of baicalin in reversing the resistance of MDRSS to azithromycin.The MICs of baicalin and azithromycin against MDRSS were detected by the serial two-fold dilution method.The synergetic effect of baicalin and azithromycin against MDRSS was determined with the checkerboard method.Then,MDRSS was sequentially passaged with the sub-MIC of baicalin.And the rates of resistance reversal and the stability of reversal were measured.The strain that recovered sensitivity to Azm was named Azithromycin-sensitive S.saprophyticus（ASSS）.Furthermore,crystal violet staining and RT-PCR assays were respectively used to measure the ability of biofilm formation and the transcript levels of msrA,mphC,and virulence-associated genes in the ASSS and MDRSS strains.Finally,mice were infected with MDRSS or ASSS to assess the therapeutic effect of azithromycin.The results showed that the MICs of baicalin and azithromycin against MDRSS were 500 and 1000 mg/L,respectively.When 125 mg/L baicalin was added,the MIC of azithromycin against MDRSS was decreased 128-fold.The value of FIC was 0.375.The sub-MIC of baicalin reversed the resistance of MDRSS to Azm.With the increase of passages,the rates of resistance reversal also increased after MDRSS was exposed to sub-MIC baicalin.The MIC of Azm against the ASSS strain was 0.488 mg/L,and it was stable within 20 passages.The ability of biofilm formation and the transcript levels of msrA,mphC,and virulence-associated genes in the ASSS strain were significantly lower than those of the MDRSS strain.Azm delayed the start time of death in mice infected with ASSS.In contrast,Azm did not have a good therapeutic effect on mice infected with MDRSS.These results indicated that baicalin had a synergetic effect with azithromycin against MDRSS and sub-MIC baicalin reversed the resistance of MDRSS to Azm.Test Ⅳ:The combination effect of baicalin and azithromycin against MDRSS infectionThe objective of this study was to explore the therapeutic effect of baicalin and azithromycin on mice infected with MDRSS.All mice were divided into 6 groups,namely,azithromycin-treated group（Azm group）,baicalin-treated group（Bac group）,azithromycin and baicalin-treated group（Azm+Bac group）,ampicillin-treated group（Amp group）,MDRSS group（SS group）,and blank control group（BC group）.Mice except the BC group were infected with MDRSS by intraperitoneal injection.After 2 h post-infection,the administration was performed once a day for 3 days.Azithromycin（75 mg/kg/d）and baicalin（6 mg/kg/d）were administered via gavage or subcutaneous injection,respectively.After 10h and 58 h post-infection,the liver,spleen,kidney,and bladder were collected to determine the bacterial burden,and a part of the kidney was fixed and stained with HE to observe the pathological changes.The blood was also collected to measure the contents of IL-6,IL-8 and TNF-α.Then sub-MIC baicalin was cocultured with MDRSS,and the transcript levels of virulence-related genes were detected by the RT-PCR method.The results showed that the survival rate of the Azm+Bac group was higher than that of the Azm,Bac and SS group（P<0.05）,but was similar to the Amp group（P>0.05）.At 10 h post-infection,the glomerulus in all mice except the BC group was atrophied,then the atrophy glomerulus was alleviated at58 h post-infection,and the glomerulus in the Azm+Bac group was similar with the BC group.At 10 h and 58 h post-infection,the contents of IL-6,IL-8,and TNF-α,and bacterial burden in organs were lower than the Bac,Azm and SS groups（P<0.05）,and were similar to those of the Amp group（P>0.05）.The sub-MIC baicalin significantly decreased the transcript levels of virulence-related genes（P<0.05）.These results indicated that baicalin and azithromycin could not treat the infection caused by MDRSS.But ampicillin or the combination of baicalin and azithromycin could treat the infection.Baicalin could inhibit the transcript levels of virulence-related genes.Test Ⅴ:The investigation of baicalin reduces the ability of biofilm formation in MDRSS by influencing agr quorum-sensing system by inhibiting the MsrA efflux pumpThis study aimed to evaluate the influences and mechanisms of baicalin on the efflux pump and biofilm formation in MDRSS,and analyze the relationship between efflux,agr system,and biofilm formation.The ethidium bromide efflux assay was used to analyze the effect of baicalin on the efflux of MDRSS.The transcript level of msrA gene in MDRSS was detected by the RT-PCR method.The content of ATP and the activity of pyruvate kinase（PK）were measured using kits.Crystal violet staining,confocal laser scanning microscopy,and scanning electron microscopy were used to analyze the influence of baicalin on biofilm formation of MDRSS.The transcript levels of msrA and agr-system associated genes were detected by RT-PCR.Pearson’s correlation analysis was used to analyze the relationship between efflux and biofilm formation,agr-system.The results showed that sub-MIC baicalin remarkably inhibited the efflux of MDRSS compared with the control group（P<0.05）.The transcript levels of msrA gene,contents of ATP and activities of PK in the baicalin group were lower than those of the MDRSS control group（SS group,P<0.05）.Baicalin significantly inhibited the ability of the biofilm formation,the transcript levels of msrA,and agr-system-related genes compared with the SS group at 24 h and 48 h（P<0.05）.The above indices in the 250 mg/L baicalin group were the same as the efflux inhibitor group（P>0.05）.Correlation analysis indicated that there was a strong positive correlation between the efflux pump and biofilm formation and the agr-system（P<0.05）.These results suggested that baicalin inhibited the efflux of MDRSS by hindering the transcript level of msrA gene,decreasing the activity of PK and the content of ATP.Baicalin inhibited biofilm formation and agr quorum-sensing system by inhibiting the MsrA efflux pump in MDRSS.Test Ⅵ:Baicalin inhibits the adhesion and aggregation phases of MDRSS biofilm formationThis investigation aimed to explore the influence and mechanisms of baicalin on primary adhesion and aggregation phases of biofilm formation.The main components and the process of biofilm formation were detected by crystal violet staining.To confirm the influence of baicalin on biofilm formation,baicalin was added at different time points.Fibrinogen binding assay and aggregation assay were used to verify the effects of baicalin on adhesion and aggregation,respectively.CLSM was used to observe the influence of baicalin on the production of surface proteins.An autolysis assay induced by Triton-X100 was used to detect the influence of baicalin on autolysis.The content of extracellular DNA（eDNA）was measured.The transcript levels of adhesion and autolysis-related genes were detected by RT-PCR.The results showed that the adhesion phase was 0-6 h,aggregation phase was 6-10h,maturation phase was 10-24 h,and dispersion phase was 24-48 h.The main components of biofilm in MDRSS were protein and eDNA.Compared with the control group,baicalin prominently decreased the ability of biofilm formation when baicalin was added before the adhesion and aggregation phases（P<0.05）.Baicalin significantly increased the relative adhesion inhibition rates and decreased the rates of aggregation（P<0.05）.Baicalin decreased the green fluorescence.The rates of cells remaining in the 250 mg/L baicalin group were higher than the MDRSS control group at every time point.And the contents of eDNA were decreased by 250 mg/L baicalin.250 mg/L baicalin downregulated the transcript levels of adhesion and autolysis-related genes.Therefore,these results indicated that baicalin inhibited the adhesion and aggregation phases of biofilm formation by influencing the production of surface proteins and cell autolysis.Test Ⅶ:The synergetic effect of baicalin and azithromycin against MDRSS in biofilm formThis study aimed to study the effect and mechanism of baicalin and azithromycin against MDRSS in biofilm form.The minimum inhibitory concentrations of baicalin and azithromycin against MDRSS in biofilm form（MICB）were detected by the serial two-fold dilution method.The synergetic effect of baicalin and azithromycin against MDRSS in biofilm form was measured using the checkerboard method.Crystal violet staining was used to detect the ability of baicalin and azithromycin to eradicate biofilm.CLSM and SEM were used to investigate the bactericidal effect.The activity of AKP and the content ofβ-GAL were also measured.RT-PCR was used to detect the transcript levels of WalK/R system-associated genes.The correlation analysis between the WalK/R system and the bactericidal index was conducted using Pearson’s method.The results showed that the MICBs of baicalin and azithromycin against MDRSS in biofilm form were 9000 and 6000 mg/L,respectively.Baicalin and azithromycin had a synergetic effect.The quantities of remaining biofilm,the ratios of PI,the activities of AKP,the contents ofβ-GAL,and transcript levels of WalK/R system associated genes in the combination of azithromycin and baicalin（Azm+Bac）group were higher than baicalin（Bac）group,azithromycin（Azm）group and MDRSS control（SS）group（P<0.05）.The rates of dispersion in the Azm+Bac group were lower than those of the other groups.In the Azm+Bac group,many bacteria were wrinkled and deformed.In the Bac group,the contents ofβ-GAL and transcript levels of WalK/R-system associated genes were higher than the SS group（P<0.05）,and few bacteria were wrinkled and deformed.Correlation analysis indicated that there was a positive correlation between the transcript levels of WalK/R system-associated genes and the bactericidal indices.These results indicated that baicalin enhanced the ability of azithromycin against MDRSS in biofilm form by increasing the permeability of cell walls and cell membranes and regulating the transcript levels of WalK/R system-related genes.Test Ⅷ:The effect of baicalin and azithromycin destroy the immature and mature MDRSS biofilm in miceTo investigate the effect of baicalin and azithromycin destroying the immature and mature MDRSS biofilm,the cutaneous mouse infection model was established.Mice in the immature biofilm group were administrated with drugs after the model was established for 3days.Immature biofilm groups were divided into azithromycin group（IAzm group）,baicalin group（IBac group）,azithromycin and baicalin group（IAzm+Bac group）,MDRSS group（ISS group）,blank control group（IBC group）.Mice in the mature biofilm groups were administrated with drugs on the 4th day after infection.Treatment groups were divided into azithromycin group（MAzm group）,baicalin group（MBac group）,azithromycin and baicalin group（MAzm+Bac group）,MDRSS group（MSS group）,blank control group（MBC group）.After 24 h of the last dose treatment,blood sample,tube and tissue surrounding the tube were collected.Then blood routine and bacteria burden were detected.The pathological changes in the tissue surrounding the tube were measured.The contents of cytokines were confirmed by ELISA kits.The results showed that the extent of swelling,pathological changes,the bacteria burden in tube and tissue,the numbers of white blood cells,neutrophils,lymphocytes,and the contents of cytokines in the IAzm+Bac group were significantly lower than those of the ISS and IAzm groups（P<0.05）.The bacteria burden,the number of neutrophils and monocytes,and the level of TNF-αin the IBac group were prominently lower than the ISS group（P<0.05）.The above indices in the MAzm+Bac group were lower than the MSS and MAzm groups（P<0.05）.The levels of TNF-αand CXCL₂ in the MBac group were similar to the MAzm+Bac group.The other indices in the MBac group were similar to the MAzm+Bac group.These results indicated that the combination of baicalin and azithromycin could destroy immature and mature biofilm in vivo.Baicalin alone could moderately inhibit biofilm formation in vivo.In conclusion,baicalin and azithromycin acted synergetic effect against MDRSS in vivo and vitro.Sub-MIC baicalin reversed the resistance of MDRSS to azithromycin.Baicalin acted effect by inhibiting efflux and biofilm formation of MDRSS.